Molting hormone receptor and method for screening ligand to the receptor

ABSTRACT

The present invention provides a completely novel molting hormone receptor, which is used for efficiently screening a substance that can be applied to a disinfestant or the like. An insect molting hormone receptor comprising the following polypeptide (a) and polypeptide (b) or (c): (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; (b) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; and (c) a polypeptide having the amino acid sequence shown in SEQ ID NO: 3.

TECHNICAL FIELD

The present invention relates to a molting hormone receptor havingability to bind to an insect molting hormone such as ecdysteroid and amethod for screening a ligand binding to the above receptor.

BACKGROUND ART

Various objects such as improvement of productivity or improvement ofthe quality of products can be achieved by pest control in agriculturalproduction sites such as fields. Thus, various studies regarding pestcontrol have been conducted. It has been desired that a disinfestantcomprising an insect growth inhibitor as a main component will bedeveloped, for example.

Molting and/or metamorphosis are characteristic phenomena of insects.Such molting and/or metamorphosis of insects are controlled by hormones.It has been known that molting and/or metamorphosis of insects arecontrolled by a steroid hormone, ecdysteroid. First, ecdysteroidsynthesized in the body of an insect binds to a heterodimer consistingof a molting hormone receptor and USP (ultraspiracle) existing in thenucleus through the cell membrane, so as to form a complex.Subsequently, this complex binds to a response sequence existingupstream of an early gene cluster, so as to induce the expression of theearly gene cluster. Thereafter, as a result of the expression of theearly gene cluster, the expression of a gene cluster associated with themolting and/or metamorphosis of the insect is promoted, and the moltingand/or metamorphosis of the insect progresses.

Thus, it has been known that a molting hormone receptor is located atthe uppermost stream of a signaling pathway during the molting and/ormetamorphosis of an insect. Accordingly, it is said that a search for asubstance inhibiting the binding of ecdysteroid to a molting hormonereceptor that is used as the aforementioned insect growth inhibitor iseffective for the development of a disinfestant.

However, a method for screening such a substance that inhibits thebinding of ecdysteroid to a molting hormone receptor has not yet beenestablished. A substance that is effective as a disinfestant could noteasily be screened. As a system for screening an insect growthinhibitor, screening systems such as a system using an insect as awhole, a system using a portion of tissues such as epidermis, or asystem using cultured cells have conventionally been known (refer toNon-Patent Documents 1 to 4). However, under the present circumstances,almost no studies have been conducted regarding the screening of aninsect growth inhibitor at a molecular level (protein level).

Non-Patent Document 1

Kenichi Mikitani Appl. Entomol. Zool. 31 (4): 531-536

A novel ecdysone responsive reportaer plasmid regulated by the5′-upstreme region of the Drosophila melanogaster (Diptera;Drosophilidae) acethylcholinesterase gene

Non-Patent Document 2

Kenichi Mikitani J. Insect Physiol. 42: (10) 937-941 October 1996Ecdysteroid receptor binding activity and ecdysteroid agonist activityat the level of gene expression are correlated with the activity ofdibenzoyl hydrazines in larvae of Bombyx mori

Non-Patent Document 3

Kenichi Mikitani BIOCHEM. BIOPHYS. RES. CO 227: (2) 427-432 Oct. 14,1996

A new nonsteroidal chemical class of ligand for the ecdysteroid receptor3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide shows apparent insectmolting hormone activities at molecular and cellular levels

Non-Patent Document 4

Trisyono A, Goodman C L, Grasela J J, et al. IN VITRO CELLULAR &DEVELOPMENTAL BIOLOGY-ANIMAL 36: (6) 400-404 June 2000

Establishment and characterization of an Ostriniia nubilalis cell line,and its response to ecdysone agonists

Thus, taking into consideration the aforementioned actual situation, itis an object of the present invention to provide a completely novelmolting hormone receptor and a method for screening a ligand bindingthereto, so as to efficiently screen a substance that can be applied toa disinfestant or the like.

DISCLOSURE OF THE INVENTION

As a result of intensive studies directed towards achieving theaforementioned object, the present inventors have discovered a novelbinding of a molting hormone to each domain of a molting hormonereceptor, thereby completing the present invention.

The present invention includes the following features.

(1) An insect molting hormone receptor comprising the followingpolypeptide (a) and polypeptide (b) or (c):

(a) a polypeptide, which has the amino acid sequence shown in SEQ ID NO:1 (EcR-DF), or a polypeptide, which has an amino acid sequencecomprising a deletion, substitution, or addition of one or more aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 1(EcR-DF), and which forms a complex with the following polypeptide (b)or (c) and is capable of binding to a molting hormone in a state whereit forms the above-described complex;

(b) a polypeptide, which has the amino acid sequence shown in SEQ ID NO:2 (USP-AE), or a polypeptide, which has an amino acid sequencecomprising a deletion, substitution, or addition of one or more aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 2(USP-AE), and which may form a complex with the above-describedpolypeptide (a); and

(c) a polypeptide, which has the amino acid sequence shown in SEQ ID NO:3 (USP-DE), or a polypeptide, which has an amino acid sequencecomprising a deletion, substitution, or addition of one or more aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 3(USP-DE), and which may form a complex with the above-describedpolypeptide (a).

(2) The insect molting hormone receptor described in (I) above, whereinthe above-described polypeptides (a), (b), and (c) are expressed inEscherichia coli.

(3) The insect molting hormone receptor described in (1) above, whereinthe above-described polypeptide (a) is allowed to express in Escherichiacoil and is then subjected to gel filtration, and thus, theabove-described polypeptide (a) has activity of binding to theabove-described polypeptide (b) or (c).

(4) A method for screening a ligand binding to a molting hormonereceptor, which comprises: a first step of allowing a test substance toact on the insect molting hormone receptor described in any one of (1)to (3) above; and a second step of measuring the binding of theabove-described complex to the above-described test substance.

(5) The method for screening a ligand binding to a molting hormonereceptor described in (4) above, wherein, in the above-described firststep, the above-described insect molting hormone receptor is mixed withthe above-described test substance, and the mixture is then reacted for30 to 90 minutes.

(6) The method for screening a ligand binding to a molting hormonereceptor described in (4) above, wherein, in the above-described firststep, the above-described insect molting hormone receptor is mixed withthe above-described test substance, and the mixture is then reacted at atemperature between 20° C. and 37° C.

(7) The method for screening a ligand binding to a molting hormonereceptor described in (4) above, wherein, in the above-described firststep, the above-described insect molting hormone receptor is mixed withthe above-described test substance, and the mixture is then reactedunder conditions where substantially no salts exist.

(8) An agent for screening a ligand binding to an insect molting hormonereceptor, which comprises the following polypeptide (a) and polypeptide(b) or (c):

(a) a polypeptide, which has the amino acid sequence shown in SEQ ID NO:1 (EcR-DF), or a polypeptide, which has an amino acid sequencecomprising a deletion, substitution, or addition of one or more aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 1(EcR-DF), and which forms a complex with the following polypeptide (b)or (c) and is capable of binding to a molting hormone in a state whereit forms the above-described complex;

(b) a polypeptide, which has the amino acid sequence shown in SEQ ID NO:2 (USP-AE), or a polypeptide, which has an amino acid sequencecomprising a deletion, substitution, or addition of one or more aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 2(USP-AE), and which may form a complex with the above-describedpolypeptide (a); and

(c) a polypeptide, which has the amino acid sequence shown in SEQ ID NO:3 (USP-DE), or a polypeptide, which has an amino acid sequencecomprising a deletion, substitution, or addition of one or more aminoacids with respect to the amino acid sequence shown in SEQ ID NO: 3(USP-DE), and which may form a complex with the above-describedpolypeptide (a).

(9) The agent for screening a ligand binding to an insect moltinghormone receptor described in (7) above, wherein the above-describedpolypeptides (a), (b), and (c) are expressed in Escherichia coli.

(10) The agent for screening a ligand binding to an insect moltinghormone receptor described in (8) above, wherein the above-describedpolypeptide (a) is allowed to express in Escherichia coli and is thensubjected to gel filtration, and thus, the above-described polypeptide(a) has activity of binding to the above-described polypeptide (b) or(c).

This specification includes part or all of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.2003-031606, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) is a block diagram schematically showing a domain structure ofEcR, and FIG. 1(b) is a block diagram schematically showing a domainstructure of USP;

FIG. 2 is a view showing the positions and sequences of primers preparedby cDNA cloning of EcR;

FIG. 3 is a view showing the positions and sequences of primers preparedby cDNA cloning of USP;

FIG. 4 is a view showing EcR recombinants with various lengths preparedin Example 2;

FIG. 5 is a view showing USP recombinants with various lengths preparedin Example 2;

FIG. 6 includes several photographs showing the results of SDS-PAGEperformed on EcR recombinants expressed in Escherichia coli;

FIG. 7 includes several photographs showing the results of SDS-PAGEperformed on USP recombinants expressed in Escherichia coli;

FIG. 8 is a photograph showing the results of Western blotting performedto confirm EcR recombinants;

FIG. 9A is a characteristic diagram showing the results obtained bymeasuring the molecular mass of an EcR recombinant using MALDI-TOF-MS;

FIG. 9B is a characteristic diagram showing the results obtained bymeasuring the molecular mass of another EcR recombinant usingMALDI-TOF-MS;

FIG. 9C is a characteristic diagram showing the results obtained bymeasuring the molecular mass of another EcR recombinant usingMALDI-TOF-MS;

FIG. 9D is a characteristic diagram showing the results obtained bymeasuring the molecular mass of another EcR recombinant usingMALDI-TOF-MS;

FIG. 9E is a characteristic diagram showing the results obtained bymeasuring the molecular mass of an EcR recombinant using MALDI-TOF-MS;

FIG. 10 is a photograph showing the results of SDS-PAGE, which areobtained by subjecting all EcR recombinants to a refolding reaction inwhich gel filtration chromatography has been applied, and confirmingthat all the EcR recombinant have been eluted at a position of exclusionlimit;

FIG. 11 is a photograph showing the results of SDS-PAGE performed on thepurified EcR recombinant;

FIG. 12 includes several photographs showing the results of SDS-PAGEperformed on each fraction obtained after affinity purification of USPrecombinants;

FIG. 13 is a block diagram showing an expression vector produced inExample 3;

FIG. 14 is a photograph showing the results of Western blotting, whichhas been carried out to confirm the presence of an EcR recombinant in asystem wherein the EcR recombinant has been allowed to express inmammalian cells;

FIG. 15 is a flow chart showing the process of an experiment in Example4;

FIG. 16 is a characteristic diagram showing the results obtained bymeasuring the ability of EcR recombinants and USP recombinants, whichhave been allowed to express in Escherichia coli, to bind to ponasteroneA;

FIG. 17 is a characteristic diagram showing the results obtained bymeasuring the ability of EcR recombinants and USP recombinants, whichhave been allowed to express in mammalian cells, to bind to ponasteroneA;

FIG. 18 is a characteristic diagram showing the results obtained byexamining the optimal molar ratio between EcR-DF and USP-AE in a bindingreaction between an EcR-DF/USP-AE complex and a ligand;

FIG. 19 is a characteristic diagram showing the results obtained byexamining the optimal reaction time, with regard to a binding reactionbetween an EcR-DF/USP-AE complex and a ligand;

FIG. 20 is a characteristic diagram showing the results obtained byexamining the optimal reaction temperature, with regard to a bindingreaction between an EcR-DF/USP-AE complex and a ligand;

FIG. 21 is a characteristic diagram showing the results obtained byexamining the optimal salt concentration, with regard to a bindingreaction between an EcR-DF/USP-AE complex and a ligand;

FIG. 22 is a characteristic diagram showing a binding curve in a bindingreaction between an EcR-DF/USP-AE complex and a ligand;

FIG. 23 is a characteristic diagram showing the results of Scatchardanalysis performed on a binding reaction between an EcR-DF/USP-AEcomplex and a ligand;

FIG. 24 is a characteristic diagram showing the results obtained byanalyzing the binding of each of ecdysone and an ecdysone agonist to anEcR-DF/USP-AE complex; and

FIG. 25 is a characteristic diagram showing the results obtained byanalyzing the binding of an EcR-DF/USP-AE complex to each of endocrinedisrupters and a female hormone.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail below.

A screening method to which the present invention is applied(hereinafter referred to simply as “the present screening method” attimes) comprises: allowing a test substance to act a complex consistingof a segment from D region to F region (hereinafter abbreviated asEcR-DF at times) of a molting hormone receptor derived from Spodopteralitura (hereinafter abbreviated as EcR at times) and a segment from Aregion to E region (hereinafter abbreviated as USP-AE at times) ofUltraspiracle derived from Spodoptera litura (hereinafter abbreviated asUSP at times) or a segment from D region to E region thereof(hereinafter abbreviated as USP-DE at times); and then measuring thebinding of the complex to the test substance; so as to evaluate theability of the test substance to bind to the molting hormone receptor(ligand ability).

As shown in FIG. 1 a, the term “molting hormone receptor” is used hereinto mean a receptor binding to a molting hormone, which comprises 6regions consisting of A/1 region, C region, D region, E region, and Fregion. Such a molting hormone receptor has been identified in insectsother than Spodoptera litura. The results of studies regarding suchinsects other than Spodoptera litura suggest that the C region of EcR isa DNA-binding region. In addition, it is suggested that the E region ofEcR is a hormone-binding region in insects other than Spodoptera litura.

Moreover, as shown in FIG. 1 b, USP is an orphan receptor, whichcomprises 5 regions consisting of A/B region, C region, D region, and Eregion, and forms a heterodimer together with a molting hormonereceptor.

First, EcR-DF used in the present screening method will be described. Anexample of EcR-DF may be a segment having the amino acid sequence shownin SEQ ID NO: 1. However, EcR-DF is not limited to the segment havingthe amino acid sequence shown in SEQ ID NO: 1. For example, EcR-DF maybe a segment, which has an amino acid sequence comprising a deletion,substitution, and/or addition of one or more amino acids with respect tothe amino acid sequence shown in SEQ ID NO: 1, and which forms a complexwith USP-AE or USP-DE and is capable of binding to ecdysone in a statewhere it forms the above complex. The term “more amino acids” is usedherein to mean, for example, 2 to 50 amino acids, preferably 2 to 20amino acids, and more preferably 2 to 10 amino acids.

Moreover, EcR-DF may be a segment comprising the D region and F regionof EcR. For example, a portion of the C region may be comprised on theN-terminal side of the D region of EcR. That is to say, an example of anamino acid sequence comprising an addition of 1 or more amino acids withrespect to the amino acid sequence shown in SEQ ID NO: 1 may be an aminoacid sequence, which comprises the D region and F region of EcR andcomprises a portion of the C region on the N-terminal side of the aboveD region. The term “a portion of the C region” is used herein to mean,for example, 1 to 20 amino acids, preferably 1 to 10 amino acids, andmore preferably 1 to 5 amino acids.

Next, a method for preparing EcR-DF will be described. In the presentscreening method, a method for preparing EcR-DF is not particularlylimited. For example, EcR-DF can be prepared as follows. That is, a cDNAlibrary of Spodoptera litura is first prepared, and cDNA encoding EcR iscloned using certain primers. Subsequently, using this EcR cDNA, thenucleotide sequence of the coding region of the EcR gene is determined.Primers for amplifying a region encoding EcR-DF are designed based onthe nucleotide sequence. Using the primers, PCR is carried out with EcRcDNA as a template, so as to amplify DNA encoding EcR-DF. Thereafter,the amplified DNA is cloned into a suitable vector, and a suitable hostis then transformed with the above vector. Thereafter, EcR-DF, which hasbeen allowed to express in the obtained transformant, is recovered, soas to prepare EcR-DF.

In the aforementioned method for preparing EcR, mRNA used for preparinga cDNA library may be extracted from Spodoptera litura at any stage suchas an imago, a larva, or a chrysalis. In addition, such mRNA may beextracted from any sites of Spodoptera litura. Specifically, total RNAcan be extracted from the fat body of a Spodoptera litura larva, andmRNA contained in the extracted total RNA can be used.

Preparation of a cDNA library from mRNA contained in total RNA can becarried out according to a common method. That is, first strand cDNA issynthesized from total RNA, and then, using this as a template, EcR cDNAcan be cloned.

As primers used for cloning of EcR cDNA, degenerate primers designedbased on sequences conserved among other lepidopters can be used.Specific examples of such degenerate primers may include 6 sets ofdegenerate primers as shown in FIG. 2. These 6 sets of degenerateprimers were named as EcR-F1 and EcR-R1; EcR-F2 and EcR-R2; EcR-F3 andEcR-R3; EcR-F4 and EcR-R4; EcR-F5 and EcR-R5; and EcR-F6 and EcR-R6.

The nucleotide sequences of these degenerate primers are shown below.EcR-F1: (SEQ ID NO: 4) 5′-CTGGCGGTIGGIATGMGNCC-3′ EcR-F2: (SEQ ID NO: 5)5′-GTCGGGATGMGICCNGARTG-3′ EcR-R1: (SEQ ID NO: 6)5-′CCCTTCGCGAAYTCNACDAT-3′ EcR-R2: (SEQ ID NO: 7)5′-TCGACGATIARYTGNACNGT-3′ EcR-F3: (SEQ ID NO: 8)5′-TCGCGTRCTYTTCTCACCTG-3′ EcR-F4: (SEQ ID NO: 9)5′-CGTRCTYTTCTCACCTGTTG-3′ EcR-R3: (SEQ ID NO: 10)5′-TTCCTCATCTTCATCCGACTCTGTGAT-3′ EcR-R4: (SEQ ID NO: 11)5′-CATCTTCATCCGACTCTGTGATTCTTC-3′ EcR-F5: (SEQ ID NO: 12)5′-CAGTAGATGATCACATGCCT-3′ EcR-F6: (SEQ ID NO: 13)5′-ATGATCACATGCCTCCCATT-3′ EcR-R5: (SEQ ID NO: 14)5′-TTYCAYCCAATAGAAACATC-3′ EcR-R6: (SEQ ID NO: 15)5′-GTCTATGAGCGTTCTCTCTCCT-3′

Using these degenerate primers, RT-PCR is carried out with the firststrand cDNA synthesized from the total RNA as a template, so as tosynthesize a cDNA fragment. Thereafter, the nucleotide sequence of thesynthesized cDNA fragment is determined, and the homology thereof withEcR of other lepidopters is then analyzed. When the above cDNA fragmentshows high homology with EcR of other lepidopters, it can be determinedthat it is EcR cDNA.

Subsequently, based on this nucleotide sequence, a specific primer(EcR-5′R) used in 5′ RACE for determining the nucleotide sequence of thefull-length cDNA of EcR, and a specific primer (EcR-3′F1) used in 3′RACE therefor, can be designed. EcR-5′R: (SEQ ID NO: 16)5′-ATCCTCCGGCAAAGGCTTTCACTTCAC-3′ EcR-3′F1: (SEQ ID NO: 17)5′-GTTCCTCGAGGAGATCTGGGACGTG-3′

Using these primers EcR-5′R and EcR-3′F1 and a primer (UPM) specific forthe anchor sequence of SMART™RACE cDNA Amplification Kit, PCR is carriedout with the first strand cDNA synthesized from the total RNA as atemplate, so as to synthesize a cDNA fragment. The nucleotide sequenceof UPM is shown below. UPM: (SEQ ID NO: 18)5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAG T-3′

Thereafter, from the nucleotide sequence of the synthesized cDNA and thenucleotide sequence of the cDNA fragment synthesized using theaforementioned degenerate primers, the nucleotide sequence of thefull-length cDNA of EcR can be determined. The nucleotide sequence ofthe full-length cDNA of EcR is shown in SEQ ID NO: 19, and the putativeamino acid sequence of EcR is shown in SEQ ID NO: 20.

In order to construct all expression vector having DNA encoding EcR-DF,first, using a primer EcR-D: 5′-CATATGGCTAGCAGGCCTGAGTGCGTGGTGCC-3′ (SEQID NO: 21) and a primer EcR-F: 5′-TTCGCAAGCTTCTAGAGCGCCGCGCTTTCCG-3′(SEQ ID NO: 46), which have been designed based on the nucleotidesequence of the full-length cDNA of EcR (SEQ ID NO: 19), PCR is carriedout with the cloned EcR cDNA as a template, so as to obtain a DNAfragment encoding EcR-DF.

Subsequently, this DNA fragment is inserted into a suitable vector thatdepends on a host, so as to construct an expression vector. In addition,when EcR-DF is allowed to express in a host, an expression vector may beconstructed such that a histidine tag is added to the N-terminus ofEcR-DF.

Herein, Escherichia coli is preferably used as a host because arelatively large amount of EcR-DF can be obtained therefrom. However,such a host is not limited to Escherichia coli, and various types ofcells that are conventionally used in a gene expression system, can beused. Examples of such cells may include: mammalian cell lines such asCOS-7 cells or CHO cells; and insect cells such as Sf-9.

When Escherichia coli is used as a host, a vector pET-28b(+) used forexpression in Escherichia coli can be used as an expression vector.Using the vector pET-28b(+) used for expression in Escherichia coli, ahistidine tag can be added to the N-terminus of EcR-DF to be expressed.

EcR-DF expressed in a host can be generated according to a commonmethod. When an Escherichia coli EL21(DE3) strain is transformed with anexpression vector formed by incorporating DNA encoding EcR-DF intopET-28b(+), and when the expression of EcR-DF is induced by 1PTG, EcR-DFforms an insoluble inclusion body. In such a case, EcR-DF can beobtained in a desired form by solubilizing the inclusion body and thenrefolding it.

Next, USP-AE and USP-DE used in the present screening method will bedescribed.

An example of USP-AE may be a segment having the amino acid sequenceshown in SEQ ID NO: 2. However, USP-AE is not limited to the segmenthaving the amino acid sequence shown in SEQ ID NO: 2. For example,USP-AE may be a segment, which has an amino acid sequence comprising adeletion, substitution, and/or addition of one or more amino acids withrespect to the amino acid sequence shown in SEQ ID NO: 2, and which mayform a complex with EcR-DF. The term “more amino acids” is used hereinto mean, for example, 2 to 200 amino acids, preferably 2 to 100 aminoacids, and more preferably 2 to 50 amino acids.

Moreover, USP-DE may be a segment having the amino acid sequence shownin SEQ ID NO: 3. However, USP-DE is not limited to the segment havingthe amino acid sequence shown in SEQ ID NO: 3. For example, USP-DE maybe a segment, which has an amino acid sequence comprising a deletion,substitution, and/or addition of one or more amino acids with respect tothe amino acid sequence shown in SEQ ID NO: 3, and which may form acomplex with EcR-DF. The term “more amino acids” is used herein to mean,for example, 2 to 20 amino acids, preferably 2 to 10 amino acids, andmore preferably 2 to 5 amino acids.

Preparation of these USP-AE and USP-DE can be carried out according tothe aforementioned method for preparing EcR-DF. In particular, 4 sets ofdegenerate primers used for cloning of USP-cDNA, specific primers usedfor 5′ RACE, and specific primers used for 3′ RACE are shown in FIG. 3.These 4 sets of degenerate primers were named as USP-F1 and USP-R1;USP-F2 and USP-R2; USP-F3 and USP-R3; and USP-F4 and USP-R4.

The nucleotide sequences of the 4 sets of degenerate primers, those ofthe specific primers used for 5′ RACE (USP-5′R1 and USP-5′R2), those ofthe specific primers used for 3′ RACE (USP-3′F1 and USP-3′F2), and thoseof primers specific for the anchor sequences of SMART™RACE cDNAAmplification Kit (UPM, NUP, RTG, and RTG-N), are shown below. USP-F1:(SEQ ID NO: 23) 5′-ATCAGAARTGTCTNGCNTGC-3′ USP-F2: (SEQ ID NO: 24)5′-ARTGTCTIGCNTGCGGNATG-3′ USP-R1: (SEQ ID NO: 25)5′-CTCGGACAGCACGCGRTCRA-3′ USP-R2: (SEQ ID NO: 26)5′-GACAGCACGCGRTCRAADAT-3′ USP-F3: (SEQ ID NO: 27)5′-CGATCGCITGGMGNTCNATG-3′ USP-F4: (SEQ ID NO: 28)5′-TCGCITGGMGITCNATGGAG-3′ USP-R3: (SEQ ID NO: 29)5′-CTACAKGATIYTGGTRTCGA-3′ USP-R4: (SEQ ID NO: 30)5′-CAKGATIYTGGTRTCGATSG-3′ USP-5′R1: (SEQ ID NO: 31)5′-TGAGCTGCTTGGATGTGCAT-3′ USP-5′R2: (SEQ ID NO: 32)5′-GCTGCTTGGATGTGCATCCT-3′ USP-3′F1: (SEQ ID NO: 33)5′-CGCTCCATCTCGCTGAAGAGCTTC-3′ USP-3′F2: (SEQ ID NO: 34)5′-GTCCATCGCGTCCTACATC-3′ UPM: (SEQ ID NO: 35)5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAG T-3′ NUP: (SEQ ID NO:36) 5′-AAGCAGTGGTAACAACGCAGAGT-3′ RTG: (SEQ ID NO: 37)5′-AACTGGAAGAATTCGCGGCCG-3′ RTG-N: (SEQ ID NO: 38)5′-TGGAAGAATTCGCGGCCGCAG-3′

The nucleotide sequence of the cDNA of USP that is cloned according tothe aforementioned method for preparing EcR-DF is shown in SEQ ID NO:39, and the putative amino acid sequence of USP is shown in SEQ ID NO:40.

DNA encoding EcR-DF is amplified by PCR using EcR cDNA as a template.Thereafter, the amplified DNA is cloned into a suitable vector, and asuitable host is then transformed with the above vector. Thereafter,EcR-DF, which has been allowed to express in the obtained transformant,is recovered, so as to prepare EcR-DF.

In order to construct an expression vector having DNA encoding USP-AE, aprimer USP-A: 5′-TTCTTGCTAGCATGTCCATAGAGTCGCGTTTAG-3′ (SEQ ID NO: 41),and a primer USP-Er1: 5′-ATTACAAGCTTACATGACGTTGGCGTCGATG-3′ (SEQ ID NO:42) are used. In order to construct an expression vector having DNAencoding USP-DE, a primer USP-D: 5′-CATATGGCTAGCAAGAGGGAGGCAGTTCAGGAG-3′(SEQ ID NO: 43), and the primer USP-Er1 (SEQ ID NO: 42) are used. Usingthese primers, USP-AE and USP-DE can be obtained as in the case ofEcR-DF, as described above.

In the present screening method, using EcR-DF, USP-AE, and USP-DE asobtained above, the ability of a test substance to bind to a moltinghormone receptor (ligand ability) is evaluated. For example, the abilityof a test substance to bind to a complex consisting of EcR-DF and USP-AEor USP-DE can be evaluated by labeling the test substance. In thisevaluation, EcR-DF and USP-AE or USP-DE may be mixed with a testsubstance, and the ability of the test substance to bind to a complexconsisting of EcR-DF and USP-AE or USP-DE may be then evaluated.Otherwise, a complex consisting of EcR-DF and USP-AE or USP-DE haspreviously been prepared, and thereafter, a test substance may beallowed to act on the complex, and the ability thereof may be evaluated.

In order to label a test substance, a method using radioisotope may beused.

As stated above, the present screening method provides an effectivemeans for searching for a substance that suppresses induction of theexpression of a gene cluster associated with molting and/ormetamorphosis due to a heterodimer of EcR and USP, using a complexconsisting of EcR-DF and USP-AE or USP-DE.

The present invention will be described more in detail below in thefollowing examples. However, the examples are not intended to limit thetechnical scope of the present invention.

EXAMPLE 1

Determination of Nucleotide Sequences of EcR and USP Derived fromSpodoptera litura

Material

In the present example, a Spodoptera litura larva (the last instar (thesixth-instar) larva (with a body length of approximately 4 cm and a bodyweight of approximately 1.5 g)) provided from Kumiai Chemical IndustryCo., Ltd. was used. The Spodoptera litura larva was excised under astereoscopic microscope, and a fat body thereof was extirpated using aforceps. It was then placed in an Eppendorf tube and then freeze-driedwith liquid nitrogen. The thus freeze-dried product was then conservedat −80° C. until it was used for extraction of total RNA.

RT-PCR

In order to extract total RNA from the extirpated fat body of Spodopteralitura, an RNA extraction reagent ISOGEN (Nippon Gene Co., Ltd.) wasused in accordance with protocols provided with the reagent. All aliquotof the extracted total RNA solution was diluted with DEPC-treated water,and the concentration of the diluted solution was quantified with aspectrophotometer. 2 μg of total RNA was prepared from the obtainedtotal RNA solution. Thereafter, first strand cDNA was synthesized fromthe total RNA using Ready-To-Go™ T-Primed First-Strand Kit (AmershamBiosciences) in accordance with protocols provided with the kit. At thesame time, first strand cDNA was synthesized from 1 μg of total RNAusing SMART™RACE cDNA Amplification Kit (CLONTECH), separately.

Using the synthesized first strand cDNA as a template, a PCR reactionwas carried out employing a thermal cycler. Primers shown in FIGS. 2 and3 were used as degenerate primers. The composition of a PCR reactionsolution is shown in Table 1. TABLE 1 PCR reaction solution 10 × PCRBuffer (TAKARA) 2 μl dNTP Mixture (2.5 mM) 2 μl Primer (forward, 10 μM)2 μl Primer (reverse, 10 μM) 1 μl Template DNA 0.5 μl   TAKARA Taq ™ (5U/μl) 0.1 μl   Sterilized water 13.5 μl   Total 20.1 μl  

The reaction was carried out by maintaining at 94° C. for 3 minutes,performing a cycle consisting of denaturation at 94° C. for 30 seconds,annealing at 55° C. for 30 seconds, and elongation at 72° C. for 30seconds, 35 times, and then treating at 72° C. for 7 minutes.

A PCR product was subcloned using TA Cloning Kit (Invitrogen). That is,a PCR product was ligated to a vector (pCR 2.1) included in the kit, andcompetent cells INVαF′ (Invitrogen) were then transformed with theobtained plasmid DNA. Thereafter, plasmid DNA into which the PCR producthad been inserted was selected, and the nucleotide sequence of the PCRproduct was determined. Thermo Sequence Cy5.5 dye terminator cyclesequencing kit (Amersham Biosciences) was used for a cycle sequencereaction in which plasmid DNA was used as a template.

5′ RACE and 3′ RACE

A primer used for 5′ RACE and a primer used for 3′ RACE were preparedbased oil the nucleotide sequence of a cDNA fragment that had beenamplified by the aforementioned RT-PCR. In addition, cDNA used as atemplate in 5′ RACE was synthesized using SMART™RACE cDNA AmplificationKit. On the other hand, cDNA used as a template in 3′ RACE wassynthesized using Ready-To-Go™ T-Primed First-Strand Kit.

Using these primers, the nucleotide sequence of the full-length cDNA ofEcR and the nucleotide sequence of the full-length cDNA of USP weredetermined, The nucleotide sequence of the full-length cDNA of EcR isshown in SEQ ID NO: 19, and the putative amino acid sequence of EcR isshown in SEQ ID NO: 20. The nucleotide sequence of the full-length cDNAof USP is shown in SEQ ID NO: 39, and the putative amino acid sequenceof USP is shown in SEQ ID NO: 40.

EXAMPLE 2

Expression of EcR Recombinant and USP Recombinant (Escherichia coli)

Preparation of DNA Fragment

First, in order to produce various recombinants with different lengthscontaining the ligand-binding region (E region) of EcR, the followingprimers, to which the sequence of a specific restriction site was added,were designed based on the nucleotide sequence obtained in Example 1:EcR-A: (SEQ ID NO: 44) 5′-TTCTTGCTAGCATGTCCATAGAGTCGCGTTTAG-3′ EcR-D:(SEQ ID NO: 21) EcR-Ef: (SEQ ID NO: 45)5′-TTTTTGGATCCACAAGAAGGCTATGAACAACC-3′ EcR-Er1: (SEQ ID NO: 22)5′-TTGTTAAGCTTAGTCCCAGATCTCCTCGAGGA-3′ EcR-F: (SEQ ID NO: 46)5′-TTCGCAAGCTTCTAGAGCGCCGCGCTTTCCG-3′

Moreover, in order to produce various recombinants with differentlengths containing the ligand-binding region (E region) of USP, thefollowing primers, to which the sequence of a specific restriction sitewas added, were designed based on the nucleotide sequence obtained inExample 1: USP-A: (SEQ ID NO: 41)5′-TTCTTGCTAGCATGTCCATAGAGTCGCGTTTAG-3′ USP-D: (SEQ ID NO: 43)5′-CATATGGCTAGCAAGAGGGAGGCAGTTCAGGAG-3′ USP-Ef: (SEQ ID NO: 47)5′-TTTTTGGATCCTTCAGTGCAGGTACAGGAATT-3′ USP-Er1: (SEQ ID NO: 42)5′-ATTACAAGCTTACATGACGTTGGCGTCGATG-3′

Using these primers, a PCR reaction was carried out with the plasmidused for cloning in Example 1 as template DNA. The obtained PCR productwas subcloned. The composition of a PCR reaction solution is shown inTable 2. TABLE 2 PCR reaction solution 10 × EX PCR Buffer (TAKARA)   3μl dNTP Mixture (2.5 mM)   3 μl Primer (forward, 10 μM)  1.5 μl Primer(reverse, 10 μM)  1.5 μl Template DNA 0.15 μl TAKARA EX Taq ™ Hot StartVersion (5 U/μl) 0.15 μl Sterilized water 20.7 μl Total   30 μl

The reaction was carried out by maintaining at 94° C. for 3 minutes,performing a cycle consisting of denaturation at 94° C. for 30 seconds,annealing at 60° C. for 30 seconds, and elongation at 72° C. for 1minute, 15 times, and then treating at 72° C. for 7 minutes.

The PCR product was recovered from agarose gel, and was then subclonedusing TOPO TA Cloning™ Kit (Invitrogen) in accordance with protocolsprovided with the kit. A DNA sequencer was used to confirm that thenucleotide sequence of a PCR product of interest was correct.Thereafter, a plasmid was digested with specific restriction enzymes (acombination of BamHI-HindIII or NheI-HindIII). The resultant wassubjected to 1% agarose gel electrophoresis again, and a DNA fragment ofinterest was then recovered from the gel.

In the present example, as shown in FIG. 4, full-length EcR (EcR-AF) wasobtained using primers EcR-A and EcR-F; a DNA fragment encoding asegment (EcR-DF) from D region to F region of EcR was obtained usingprimers EcR-D and EcR-F; a DNA fragment encoding a segment (EcR-DE) fromD region to E region of EcR was obtained using primers EcR-D andEcR-Er1; a DNA fragment encoding a segment (EcR-EF) from E region to Fregion of EcR was obtained using primers EcR-Ef and EcR-F; and a DNAfragment encoding a segment (EcR-E) corresponding to the E-region of EcRwas obtained using primers EcR-Ef and EcR-Er1. Moreover, in the presentexample, as shown in FIG. 5, a DNA fragment encoding a segment (USP-AE)from A region to E region of USP was obtained using primers USP-A andUSP-Er1; a DNA fragment encoding a segment (USP-DE) from D region to Eregion of USP was obtained using primers USP-D and USP-Er1; and a DNAfragment encoding a segment (USP-E) corresponding to the E region of USPwas obtained using primers USP-Ef and USP-Er1.

Transformation

In order to allow an EcR recombinant and a USP recombinant encoded bythe obtained DNA fragments to express in Escherichia coli, an expressionvector used for transformation was first constructed. As a plasmidvector, pET-28b(+) (Novagen) was used. This vector was constructed suchthat a histidine (His) tag was added to the N-terminus of a protein ofinterest. This plasmid DNA was prepared from 25 ml of a culturesolution, using High Purity Plasmid Midiprep System (MARLINGENBIOSCIENCE). Whether or not insert DNA of interest had been preciselyinserted into a vector with no displacements in the triplet codon andthe frame was confirmed, and it was then used as an expression vectorfor the following experiments.

Subsequently, using the thus constructed expression vector and thecompetent cells of an Escherichia coli DH5α strain, the cells weretransformed with the vector according to the heat shock method andelectroporation.

Expression of Protein of Interest

A single colony of the Escherichia coli BL21(DE3) strain containing theabove expression vector was inoculated into 5 ml of an LB medium, and itwas then subjected to shake culture at 37° C. overnight (preculture).Thereafter, a 500-ml Erlenmeyer flask or Sakaguchi flask equipped with abaffle was used as an incubator, and 100 ml of an LB medium and 100 μlof kanamycin were added thereto. Thereafter, 5 ml of the preculturesolution was further added thereto. The obtained mixture was subjectedto shake culture at 37° C. (culture). When OD₆₀₀ became 0.6 to 0.8, IPTGwas added thereto, so as to initiate the induction of a protein ofinterest. Conditions for induction of each construct are shown in Table3. It is to be noted that the same conditions were applied to EcR-AF,EcR-DF, EcR-DE, EcR-EF, and EcR-E. TABLE 3 IPTG final concentrationInduction temperature Time EcR   1 mM   37° C. 3 h USP-AE 0.1 mM   20°C. 16 h  USP-DE 0.2 mM 26.5° C. 6 h USP-E 0.5 mM 26.5° C. 4 h

After the induction, the culture solution was centrifuged at 4° C. at8,000 rpm for 5 minutes, so as to collect cells. In the case of an EcRrecombinant, cells were well suspended in 15 ml of PBS. In the case of aUSP recombinant, cells were well suspended in 10 ml of a lysis buffer(50 nM NaH₂PO₄/300 mM NaCl/10 mM imidazole (pH8.0)). Thereafter, 10 mgof lysozyme (SIGMA) was added to each suspension, and the obtainedmixture was slowly suspended with a rotator in a low-temperature chamberfor 1 hour. It was then freeze-dried at −80° C. After 1 hour or more hadpassed, the freeze-dried product was unfrozen. Thereafter, using anultrasonic disintegrator, cells (the USP recombinant) were disintegratedat 120 W for 5 minutes, and cells (the EcR recombinant) weredisintegrated at 120 W for 10 minutes. Thereafter, the resultant wascentrifuged at 4° C. at 10,000 rpm for 15 minutes.

In the case of the EcR recombinant, a precipitate fraction obtainedafter centrifugation was suspended in 4 ml of PBS. Thereafter, 1 ml of25% Triron X-100 was added thereto, and the obtained mixture was thenslowly suspended with a rotator at room temperature for 4 hours toovernight. Subsequently, the suspension was centrifuged at 4° C. at9,000 rpm for 10 minutes. The obtained precipitate was centrifuged in 4ml of PBS at 4° C. at 9,000 rpm for 5 minutes and then washed. Thisoperation was repeated 5 to 10 times to eliminate Triton X-100 (untilthe precipitate lost stickiness). Finally, the supernatant wascompletely eliminated, and the remaining precipitate was then conservedat −20° C. until it was used for purification. On the other hand, in thecase of the USP recombinant, cells were disintegrated, and thesupernatant fraction obtained after centrifugation was used for thesubsequent purification.

Subsequently, as a result of confirmation by SDS-PAGE, it was found thatthe expressed EcR recombinants (EcR-AF, -DF, -DE, -EF, and -E) existedin an insoluble inclusion body fraction (FIG. 6). On the other hand, inthe case of all the expressed USP recombinants (USP-AE, -DE, and -E), aportion thereof was recovered as a soluble fraction (FIG. 7).

SDS-PAGE was carried out as follows. A sample was mixed with anappropriate amount of sample buffer (0.2M Tris-HCl (pH6.8)/8% SDS/24%β-ME/40% glycerol/0.05% BPB), and the obtained mixture was treated at100° C. for 5 minutes. As a molecular weight marker, LMW Calibration Kitfor SDS Electrophoresis (Amersham Biosciences) was used. Aftercompletion of the electrophoresis, the gel was fixed with a decolorizingsolution (50% methanol/7% acetic acid) for 15 minutes, and it was thenshaken in a CBB staining solution (0.25% CBB R-250/50% methanol/5%acetic acid) for 15 minutes. Finally, decolorization was carried outwith a decolorizing solution until the color of background disappeared.

In order to confirm that the expressed recombinant was the one ofinterest, Western blotting was carried out using an anti-His-tagantibody. Precision Prestained Standard (BIO-RAD) was used as amolecular weight marker. After completion of the electrophoresis, usinga blocking device, the gel was blocked on a PVDF membrane (ATTO) at 100mA for 30 minutes. After completion of the blocking, in order to preventnon-specific adsorption of the antibody, blocking was carried out inTBST (137 mM NaCl/2.68 mM KCl/25 mM Tris/0.05% Tween-20) containing 5%skimmed milk for 2 hours to overnight. After completion of the blocking,the membrane was washed with TBST for 10 minutes 4 times. Subsequently,the membrane was incubated at room temperature in an anti-His-antibody(Amersham Biosciences) that had been 2,000 times diluted with TBST. 1.5hours later, the membrane was washed with TBST for 10 minutes 4 times.Thereafter, an anti-mouse IgG conjugated HRP that had been 5,000 timesdiluted with TBST was added thereto, and the obtained mixture wasincubated at room temperature. 1 hour later, the membrane was washedwith TBST for 10 minutes 4 times. SuperSignal West Dura ExtendedDuration Substrate (Pierce) was then added thereto as a substrate ofHRP, so that the resultant was allowed to emit chemoluminescence. Theemitted chemoluminescence was then analyzed with an imaging analyzer. Asa result, as shown in FIG. 8, it was found that all the expressionproducts were those of interest. Thereafter, the sample subjected toSDS-PAGE was cut out of the gel, and the molecular mass of a digestobtained by digestion with trypsin was measured using MALDI-TOF-MS. AsShown in FIGS. 9A, 9B, 9C, 9D, and 9E, digests of the products ofinterest were confirmed. Thus, it was found that all the obtainedexpression products were products of interests.

Purification of Protein Of Interest

(EcR Recombinant)

As stated above, all the EcR recombinants form insoluble inclusionbodies, and expression products exist in such inclusion bodies. Sincesuch an EcR recombinant cannot directly be used for the subsequentexperiment regarding binding to a ligand, which will be described later,it should be solubilized and refolded, so that it can bind to a ligand.In such a refolding reaction, it is necessary to solubilize an inclusionbody in a high-concentration urea or a guanidine hydrochloride aqueoussolution and then gradually eliminate such urea or guanidinehydrochloride. Thus, dialysis or dilution has often been applied. In thepresent example, an EcR recombinant was first solubilized in 8 M urea,and it was then subjected to a refolding reaction in which dilution wasapplied. When the final concentration of urea became 1 M or less, thesolubilized protein was reprecipitated, and thus it becameinsolubilized. Thus, refolding was attempted using gel filtrationchromatography. The aim of this method is that, using gel with a smallexclusion limit, the EcR recombinants that cannot be incorporated intothe pores of the gel are eluted to an exclusion limit position. On theother hand, since urea has a low molecular weight, it can beincorporated into the pores of the gel, and thus it is eluted later thanthe EcR recombinants are. Thus, it was considered that urea can beeliminated from the solubilized EcR recombinant-urea solution, and thatthe EcR recombinants can be recovered in a solubilized state. Such arefolding reaction was carried out applying gel filtrationchromatography. As a result, it was confirmed by SDS-PAGE that all theEcR recombinants were eluted to an exclusion limit position (FIG. 10).

Each of the eluted EcR recombinants was concentrated by ultrafiltration.The concentration of this sample was quantified by the Bradford method.As a result, it was found that 186 μg of EcR-AF, 206 μg of EcR-DF, 98 μgof EcR-DE, 46 μg of EcR-EF, and 182 μg of EcR-E were obtained from 25 mleach of the culture solution. The thus purified EcR recombinant solutionwas subjected to SDS-PAGE. The results are shown in FIG. 11.

(USP Recombinant)

As stated above, several USP recombinants existed in a soluble fraction(refer to FIG. 7). Since a His-tag was added to the N-terminus of such aUSP recombinant, the USP recombinant was subjected to affinitypurification using a nickel resin (FIG. 12). It was concentrated byultrafiltration, and was then quantified by the Bradford method. As aresult, it was found that 814 μg of USP-AE, 417 μg of USP-DE, and 1.3 mgof USP-E were obtained from 100 ml each of the culture solution.

EXAMPLE 3

Expression of EcR Recombinant and USP Recombinant (Cultured AnimalCells)

In order to use as positive controls in Example 4 “Analysis of abilityof EcR recombinant and USP recombinant to bind to ligand” describedlater, ill Example 3, the EcR recombinant and the USP recombinant wereallowed to express in cultured animal cells, and extracts were preparedfrom the cells.

COS-7 cells were used as cultured animal cells. As an expression plasmidused herein, EcR-AF, EcR-DF, USP-AE, or USP-DE was inserted into aposition downstream of an SRα promoter, and a FLAG tag was then added tothe N-terminus thereof (FIG. 13).

ECR-AF incorporated into this expression plasmid could be obtained byPCR using the plasmid used for cloning in Example 1 as a template andusing a set of primers, EcR-Af and EcR-Fr. EcR-DF incorporated into theexpression plasmid could be obtained by PCR using the plasmid used forcloning in Example 1 as a template and using a set of primers, EcR-Dfand EcR-Fr. The nucleotide sequences of the primers EcR-Af, EcR-Df, andEcR-Fr are shown below. EcR-Af: (SEQ ID NO: 48)5′-CATTAGGATCCATGTCCATAGAGTCGCGTTTAG-3′ EcR-Df: (SEQ ID NO: 49)5′-CATTAGGATCCAGGCCTGAGTGCGTGGTGCCT-3′ EcR-Fr: (SEQ ID NO: 50)5′-GATTTACTAGTCTAGAGCGCCGCGCTTTCCG-3′

USP-AE incorporated into the expression plasmid could be obtained by PCRusing the plasmid used for cloning in Example 1 as a template and usinga set of primers, USP-Af and USP-Fr. USP-DE incorporated into theexpression plasmid could be obtained by PCR using the plasmid used forcloning in Example 1 as a template and using a set of primers, USP-Dfand USP-Er. The nucleotide sequences of the primers USP-Af, USP-Df, andUSP-Er are shown below. USP-Af: (SEQ ID NO: 51)5′-ATAACGGATCCATGTCAGTGGCGAAGAAAGATAAG-3′ USP-Df: (SEQ ID NO: 52)5′-ATTACGGATCCAAGAGGGAGGCAGTTCAAGAG-3′ USP-Er: (SEQ ID NO: 53)5′-ATTACACTAGTTACATGACGTTGGCGTCGATG-3′

Each expression plasmid DNA was independently introduced into COS-7cells. 48 hours later, each extract was prepared from the cells. Inaddition, plasmid DNA into which EcR-AF had been inserted and plasmidDNA into which USP-AE had been inserted were co-introduced into COS-7cells, and an extract was then prepared in the same manner. Moreover,plasmid DNA into which, EcR-DF had been inserted and plasmid DNA intowhich USP-AE had been inserted were co-introduced into COS-7 cells, andan extract was then prepared in the same manner. Furthermore, plasmidDNA into which EcR-AF had been inserted and plasmid DNA into whichUSP-DE had been inserted were co-introduced into COS-7 cells, and anextract was then prepared in the same manner. Further, plasmid DNA intowhich EcR-DF had been inserted and plasmid DNA into which USP-DE hadbeen inserted were co-introduced into COS-7 cells, and an extract wasthen prepared in the same manner. The expression of a product ofinterest in the COS-7 cells was confirmed by Western blotting using ananti-FLAG antibody.

By the aforementioned operations, 4 types of extracts containing each ofEcR-AF, EcR-DF, USP-AE, and USP-DE, an extract containing thecoexpressed EcR-AF and USP-AE, an extract containing the coexpressedEcR-DF and USP-AE, an extract containing the coexpressed EcR-AF andUSP-DE, and an extract containing the coexpressed EcR-DF and USP-DE,were prepared.

Western blotting using an anti-FLAG antibody was performed on theobtained extracts, so as to confirm the expression of proteins ofinterest. The results are shown in FIG. 14. As shown in FIG. 14, thesingle expression and coexpression of proteins of interest wereconfirmed in all of the extracts.

EXAMPLE 4

Analysis of Ability of EcR Recombinant and USP Recombinant to Bind toLigand

In Example 4, using the EcR recombinants and USP recombinants obtainedin Examples 2 and 3, the ability of complexes consisting of these EcRrecombinants and USP recombinants to bind to a ligand was analyzed. Inthe present example, ponasterone A, an ecdysone agonist derived fromplants, was used as a ligand.

In Example 4, in order to separate a binding form from a free form, thecharcoal dextran method was applied. This is a common method appliedwhen a steroid hormone is used as a ligand. By adding an activatedcarbon solution coated with dextran to a reaction solution when thereaction is terminated, a ligand that has not bound to a receptor isadsorbed on the activated carbon. Thereafter, the reaction solution iscentrifuged, thereby separating an activated carbon fraction(precipitate fraction) from a supernatant fraction in which areceptor/ligand complex exists.

The flow of the experiment in Example 4 is shown in FIG. 15. In order toprevent the non-specific binding of ponasterone A to a receptor (acomplex consisting of an EcR recombinant and a USP recombinant), 1% BSAwas first added to a binding buffer (20 mM HEPES (pH7.4)/5 mM DTT/1 nMPMSF). Thereafter, USP-AE or USP-DE was mixed with the EcR-AF, EcR-DF,EcR-DE, EcR-EF, or EcR-E, purified in Example 2. Thereafter,[³H]ponasterone A ([24, 25, 26, 27-3H] Ponasterone A (AmericanRadiolabeled Chemicals Inc.)) with a final concentration of 1.37 nM wasfurther added thereto. The obtained mixture was reacted at 25° C. 40minutes later, the reaction was terminated by addition of 200 μl of anactivated carbon solution (0.5% Charcoal, dextran coated (SIGMA)/20 mmHEPES/50 mM NaCl). A supernatant fraction obtained after centrifugationwas mixed with a liquid scintillator, and the radioactivity (DPM) of theobtained mixture was measured using a liquid scintillation counter(ALOKA LSC-5100). The obtained measurement value was defined as thetotal binding amount. Moreover, the same experiment was carried out withthe exception that nonradioactive ponasterone A that was 10,000 timesstronger than [³H]ponasterone A was added. Then, the amount of a ligandnon-specifically binding to a receptor was obtained. The amount of aligand specifically binding to a receptor was defined as a valueobtained by subtracting the non-specific binding amount from the totalbinding amount.

The results are shown in FIG. 16. FIG. 16 shows that a complexconsisting of EcR-DF and USP-AE and a complex consisting of EcR-DF andUSP-DE had ability to bind to a ligand. On the other hand, in the singleuse of EcR or USP, no specific bindings were observed regardless of thelength of EcR or USP.

At the same time, the same binding experiment was carried out using theextracts prepared in Example 3. As a result, as shown in FIG. 17, whenEcR-DF and USP-AE, or EcR-DF and USP-DE, were allowed to coexpress incells, a strong binding to a ligand was observed (lanes 7 and 8).However, when each of the above combinations was allowed to singlyexpress and they were then mixed with each other, no such bindings wereobserved (lanes 3 and 4). Moreover, when EcR-AF was combined with USP-AEor when EcR-AF was combined with USP-DE, no bindings to a ligand wereobserved both in the case of coexpression and in the case of mixing themafter a single expression.

From the above results, it was found that since EcR-DF binds toponasterone A, not only the region E, but also at least both the Dregion and F region should bind to ponasterone A. It has been suggestedso far that the D region is associated with dimerization with USP. Theresults obtained in the present example strongly support thatdimerization is essential for binding to a ligand. With regard to the Fregion, the length of the amino acids thereof is extremely short,conservativeness among various organisms is low, and the role of thestructural chemical activity thereof is unclear under the presentcircumstances. It was at least found that the F region plays animportant role for binding to a ligand.

In both an expression system using Escherichia coli and an expressionsystem using mammalian cells, the binding of the full-length EcRrecombinant (EcR-AF) to ponasterone A was observed, but it was extremelyweak. Accordingly, in the present example, it was found for the firsttime that when a ligand binding to EcR is screened, it is necessary notto use the full-length EcR, but to use an EcR-DF recombinant.

EXAMPLE 5

Analysis of Conditions for Binding Experiment

Conditions such as molar ratio, reaction time, reaction temperature, andsalt concentration are considered to be important for the interactionbetween a complex and a ligand. In order to more efficiently observe thebinding of the aforementioned complex consisting of EcR-DF and USP-AE toa ligand, such conditions as molar ratio, reaction time, reactiontemperature, and salt concentration will be analyzed in the Example 5.

Analysis of Molar ratio Between EcR-DF and USP-AE

The optimal reaction time for a binding reaction between a complexconsisting of EcR-DF and USP-AE and a ligand was analyzed as follows.First, USP-AE with various types of concentrations was added to acertain amount of EcR-DF, and the obtained mixture was then reacted at25° C. for 40 minutes in a water bath. The concentration of ponasteroneA in the reaction solution was set at 1.34 nM. The results obtained bymeasuring the binding of ponasterone A to the complex are shown in FIG.18. As is clear from FIG. 18, when the ratio between EcR-DF and USP-AEwas approximately 1:1, the binding of ponasterone A to the EcR-DF/USP-AEcomplex became saturated. From these results, it is said that the ratiobetween EcR-DF and USP-AE is preferably set at 1:1, at a molar ratio,for the binding reaction of the EcR-DF/USP-AE complex to a ligand. It isto be noted that EcR-DF and USP-AE were used at a molar ratio of 1:1 inthe subsequent experiments.

Analysis of Reaction Time

The optimal reaction time for a binding reaction between a complexconsisting of EcR-DF and USP-AE and a ligand was analyzed as follows.First, EcR-DF was mixed with USP-AE at a molar ratio of 1:1, and theobtained mixture was then reacted at 25° C. in a water bath for a periodof time of 5 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 120minutes, and 240 minutes. The concentration of ponasterone A in thereaction solution was set at 1.34 nM. The results are shown in FIG. 19.As is clear from FIG. 19, the binding of ponasterone A to the abovecomplex became almost saturated in the reaction for 60 minutes. Fromthese results, it is said that the reaction time is preferably setbetween 30 and 90 minutes for the binding reaction of the EcR-DF/USP-AEcomplex to a ligand. It is to be noted that the reaction time was set at60 minutes in the subsequent experiments.

Analysis of Reaction Temperature

The optimal reaction temperature for a binding reaction between acomplex consisting of EcR-DF and USP-AE and a ligand was analyzed asfollows. The same binding reaction as described in the above section“analysis of reaction time” was carried out with the exception that thereaction was carried out in a low-temperature chamber at 4° C. Theresults are shown in FIG. 20. As is clear from FIG. 20, the timenecessary for saturation of the binding was 8 hours in thelow-temperature chamber at 4° C. In contrast, as shown in FIG. 20, thetime necessary for saturation of the binding was 60 minutes at areaction temperature of 25° C. From these results, it is said that thereaction temperature is preferably set between 20° C. and 37° C. for thebinding reaction of the EcR-DF/USP-AE complex to a ligand. It is to benoted that the reaction temperature was set at 25° C. in the subsequentexperiments.

Analysis of Salt Concentration

The optimal salt concentration for a binding reaction between a complexconsisting of EcR-DF and USP-AE and a ligand was analyzed as follows.First, EcR-DF was mixed with USP-AE at a molar ratio of 1:1, and theobtained mixture was then reacted at 25° C. in a water bath. During thisreaction, the salt (NaCl) concentration was set at final concentrationsof 0 mM, 50 mM, 100 mM, 300 mM, and 500 mM in the reaction solution. Theresults are shown in FIG. 21. As is clear from FIG. 21, as the saltconcentration increased, the specific binding of a ligand to the abovecomplex decreased. From these results, it is said that the saltconcentration is preferably set between 0 and 100 mM for the bindingreaction of the EcR-DF/USP-AE complex to a ligand. It is to be notedthat no salts were added to the reaction solution in the subsequentexperiments.

EXAMPLE 6

Scatchard Analysis of Binding of Complex to Ligand

In Example 6, using EcR-DF and USP-AE, the dissociation constant (Kd) ofthe complex to ponasterone A was obtained as follows. First, EcR-DF wasmixed with USP-AE at a molar ratio of 1:1, and [³H]ponasterone A withvarious concentrations was then added to the mixture. An activatedcarbon solution was added to the reaction solution to eliminate freeligands. The amount of the EcR-DF/USP-AE complex binding to[³H]ponasterone A (total binding amount) was obtained by measuring theradioactivity of a supernatant fraction obtained after centrifugation.Moreover, the same experiment was carried out with the exception thatnonradioactive ponasterone A that was 10,000 times stronger than[³H]ponasterone A was added, so as to obtained non-specific binding. Theresults obtained by plotting the binding curve based on the measurementresults are shown in FIG. 22. Scatchard analysis was carried out basedon the values in the binding curve as shown in FIG. 22. The results areshown in FIG. 23. In FIG. 23, the horizontal axis indicates the specificbound amount of [³H]ponasterone A (nM), the vertical axis indicatesspecific binding amount (bound)/total binding amount-specific bindingamount (free).

As is clear from FIG. 23, as a result of the Scatchard analysis, asingle line was obtained, and Kd=2.79 nM and Bmax=0.17 nM.

EXAMPLE 7

Construction of Screening System for Insect Inhibitor

In Example 7, a screening system used for screening an insect inhibitorwas constructed based on the results of Examples 1 to 6. As described inExamples 1 to 6, when binding analysis was carried out using ponasteroneA as an ecdysone agonist, ponasterone A binding to a complex consistingof EcR-DF and USP-AE was observed. In order to confirm whether or notthis system functions as a screening system, the binding of the complexto other ecdysone agonists should also be observed. Thus, using20-hydroxyecdysone that is an ecdysone active in living insect bodiesand tebufenozide (RH-5992) that is a synthetic ecdysone agonist, thebinding experiment was carried out between the EcR-EF/USP-AE complex andeach of the above compounds.

First, EcR-DF and USP-AE were mixed into a binding buffer, and[³H]ponasterone A was then added thereto in the same manner as inExample 4. Also, the same experiment was carried out with the exceptionthat 20-hydroxyecdysone or tebufenozide that was 10,000 times strongerthan [³H]ponasterone A was added thereto, so as to obtain nonspecificbinding. As a result, both 20-hydroxyecdysone and tebufenozide exhibitedactivity of binding to the EcR-EF/USP-AE complex. Thus, the sameexperiment was carried out while the concentration of 20-hydroxyecdysoneor tebufenozide was changed. The results are shown in FIG. 24. As isclear from FIG. 24, it was found that both 20-hydroxyecdysone andtebufenozide concentration-dependently inhibit the binding of[³H]ponasterone A to the above complex.

From these results, it was found that the binding experiment usingEcR-DF and USP-AE can be used as a screening system for screening aninsect growth inhibitor.

Subsequently, in the present example, using a screening system in whichability to bind to the EcR-DF/USP-AE complex is used as an index, 4types of compounds were evaluated in terms of such binding ability. Ascompounds to be evaluated, 4-nonylphenol, stilbestrol, and diethylphthalate (endocrine disrupters), and estradiol-17β (female hormone)were used.

The results are shown in FIG. 25. As is clear from FIG. 25,4-nonylphenol, stilbestrol, and estradiol-170 exhibited activity ofbinding to the EcR-DF/USP-AE complex. In particular, 4-nonylphenol andstilbestrol exhibited strong binding activity. It has been known so farthat EcR basically binds to compounds having a steroid skeleton, such asponasterone A. On the other hand, compounds having no steroid skeletonsto which EcR binds have been limited to several types. All thesubstances exhibiting binding activity in the present example have nosteroid skeletons, and further, the fact that the substance binds to EcRhas not yet been known. From these results, it is said that a screeningsystem, in which EcR-DF and USP-AE or USP-DE expressed in Escherichiacoli are used, could be established as a novel system for searching foran insect growth inhibitor.

INDUSTRIAL APPLICABILITY

As stated above in detail, the present invention provides a completelynovel molting hormone receptor capable of binding to an insect moltinghormone. In addition, according to the present invention, a substancethat can be applied to a disinfectant or the like can efficiently bescreened using the above molting hormone receptor.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

1. An insect molting hormone receptor comprising the followingpolypeptide (a) and polypeptide (b) or (c): (a) a polypeptide, which hasthe amino acid sequence shown in SEQ ID NO: 1, or a polypeptide, whichhas an amino acid sequence comprising a deletion, substitution, oraddition of one or more amino acids with respect to the amino acidsequence shown in SEQ ID NO: 1, and which forms a complex with thefollowing polypeptide (b) or (c) and is capable of binding to a moltinghormone in a state where it forms the complex; (b) a polypeptide, whichhas the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide,which has an amino acid sequence comprising a deletion, substitution, oraddition of one or more amino acids with respect to the amino acidsequence shown in SEQ ID NO: 2, and which may form a complex with thepolypeptide (a); and (c) a polypeptide, which has the amino acidsequence shown in SEQ ID NO: 3, or a polypeptide, which has an aminoacid sequence comprising a deletion, substitution, or addition of one ormore amino acids with respect to the amino acid sequence shown in SEQ IDNO: 3, and which may form a complex with the polypeptide (a).
 2. Theinsect molting hormone receptor according to claim 1, wherein thepolypeptides (a), (b), and (c) are expressed in Escherichia coli.
 3. Theinsect molting hormone receptor according to claim 1, wherein thepolypeptide (a) is allowed to express in Escherichia coli and is thensubjected to gel filtration, and thus, the polypeptide (a) has activityof binding to the polypeptide (b) or (c).
 4. A method for screening aligand binding to a molting hormone receptor, which comprises: a firststep of allowing a test substance to act on the insect molting hormonereceptor according to any one of claims 1 to 3; and a second step ofmeasuring the binding of the complex to the test substance.
 5. Themethod for screening a ligand binding to a molting hormone receptoraccording to claim 4, wherein, in the first step, the insect moltinghormone receptor is mixed with the test substance, and the mixture isthen reacted for 30 to 90 minutes.
 6. The method for screening a ligandbinding to a molting hormone receptor according to claim 4, wherein, inthe first step, the insect molting hormone receptor is mixed with thetest substance, and the mixture is then reacted at a temperature between20° C. and 37° C.
 7. The method for screening a ligand binding to amolting hormone receptor according to claim 4, wherein, in the firststep, the insect molting hormone receptor is mixed with the testsubstance, and the mixture is then reacted under conditions wheresubstantially no salts exist.
 8. An agent for screening a ligand bindingto an insect molting hormone receptor, which comprises the followingpolypeptide (a) and polypeptide (b) or (c): (a) a polypeptide, which hasthe amino acid sequence shown in SEQ ID NO: 1, or a polypeptide, whichhas an amino acid sequence comprising a deletion, substitution, oraddition of one or more amino acids with respect to the amino acidsequence shown in SEQ ID NO: 1, and which forms a complex with thefollowing polypeptide (b) or (c) and is capable of binding to a moltinghormone in a state where it forms the complex; (b) a polypeptide, whichhas the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide,which has an amino acid sequence comprising a deletion, substitution, oraddition of one or more amino acids with respect to the amino acidsequence shown in SEQ ID NO: 2, and which may form a complex with thepolypeptide (a); and (c) a polypeptide, which has the amino acidsequence shown in SEQ ID NO: 3, or a polypeptide, which has an aminoacid sequence comprising a deletion, substitution, or addition of one ormore amino acids with respect to the amino acid sequence shown in SEQ IDNO: 3, and which may form a complex with the polypeptide (a).
 9. Theagent for screening a ligand binding to an insect molting hormonereceptor according to claim 8, wherein the polypeptides (a), (b), and(c) are expressed in Escherichia coli.
 10. The agent for screening aligand binding to an insect molting hormone receptor according to claim8, wherein the polypeptide (a) is allowed to express in Escherichia coliand is then subjected to gel filtration, and thus, the polypeptide (a)has activity of binding to the polypeptide (b) or (c).